Ceramide glycanase

The same principle described above to monitor endo-peptidase activity was used by Matsuoka to monitor a ceramide glycanase... 

Matsuoka, K., Nishimura, S. I. and Lee, Y. C. (1995). A bi-fluorescence-labeled substrate for ceramide glycanase based on fluorescence energy-transfer. Carbohydr. Res. 276, 31-42. 

Nishimura and Yamada [10-11] introduced a water-soluble polymeric support having a linker recognized by ceramide glycanase for a synthesis of ganghoside GM3 (17). Synthesis of the polymerizable lactose derivative (14) with a ceramide glycanase sensitive linker is shown in Scheme 10.3. The lactosyl ceramide (Lac-Ger) mimetic glycopolymer (15) is obtained from the monomeric precursor (14) by co-polymerization with acrylamide. 

Scheme 10.3 Ceramide glycanase mediated release by transglycosylation. a) Pd/C, MeOH b) (8), EEDQ, EtOH-CgHe c) MeONa d) CH2=CHCONH2, TMEDA, APS, DMSO-H2O, 50"C, CMP-NeuAc, a-2,3-sialyltransferase, BSA, MnCb, CIAP, 50 nM sodium cacodylate buffer, pH 7.49, ceramide, Triton CF-54, sodium citrate buffer, pH 6.0, 37°C, 61%.

Fig. 12. Transglycosylation with ceramide glycanase from American leech.

The endoglycosidase, ceramide glycanase, is used to transfer an oligosaccharide group from a water-soluble polymer to ceramide. This enzymatic tool provides an efficient new method for polymer-supported synthesis of glycosphingolipids (119). 

Figure 18-28. Ceramide glycanase mediated release by transglycosylation.

Purification of 26 from the enzyme and excess sugar building block by gel-filtration chromatography was facilitated by the presence of the poly(acryla-mide) support. Subsequent treatment of 26 with leech ceramide glycanase in the presence of an excess of ceramide as an acceptor generated the transglyco-sidated product GM3 27 in 61% yield. Thus, this polymer-supported enzyme approach afforded the glycolipid GM3 in 56% yield from the readily available precursor/monomer 24, a remarkable improvement in both the ease of synthesis and overall yield when compared to that of chemical synthesis (19). 

Nishimura, S.-l. and Yamada, K. (1997) Transfer of ganglioside GM3 oligosaccharide from a water soluble polymer to ceramide by ceramide glycanase. A novel approach for the chemical-enzymatic synthesis of glycosphingolipids. J. Am. Chem. Soc. 119,10555-10556. 

The enzyme ceramide-glycanase can be used to cleave the oligosaccharide head group from glycosphingolipids to produce glycans with free reducing termini [11]. 

S. Dastgheib, Ph.D. Thesis, University of Notre Dame, 1997, Characterization of P(l-3) galactosyltransferase and ceramide glycanase involved in biosynthesis and degradation of gangliosides. 

S.-C. Li, R. DeGasperi, J.E. Muldrey and Y.-T. Li, A unique glycosphingolipid-splitting enzyme (ceramide-glycanase from leech) cleaves the linkage between the oligosaccharide and the ceramide. Biochem. Biophys. Res. Commun., 1986,141, 346 352. 

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